An important goal of recombinant DNA technology is to obtain efficient expression of the cloned DNA. The cloning vector, widely used in molecular biology, is a small piece of DNA molecule, in which a foreign DNA fragment may be inserted. The cloning vector may be used as vehicle to transfer foreign genetic material into a cell. Insertion of the foreign DNA fragment into the cloning vector is usually carried out by (1) digesting both the vector and the foreign DNA with restriction enzyme; and (2) ligating the restriction enzyme digested fragments together. Vectors can be used for controlled expression of particular genes, with promoter sequence to drive transcription of the transgene cloned in the vector.
Once the vector is inside the cell, the protein that is encoded by the transgene is produced by cellular transcription and translation. After the expression of the gene product, the resulted protein of interest needs to be purified and isolated from other proteins of the host cell. To facilitate the purification and/or isolation process, the cloned transgene usually has a tag, such as histidine (His) tag. In addition, GFP (green fluorescent protein) sequence is often used as biomarker to follow the expression process. In cells where the tagged transgene is expressed, the GFP is also produced, and those cells can be observed under fluorescence microscopy and isolated by FACS.
Enzymes are proteins that catalyze chemical reactions. Almost all processes in biological cells need enzymes. Enzymes are widely used in the chemical industry and other industrial applications. For example, enzymes can be applied in the fermentation industry as food additives, and are also commonly used in food processing and in the production of food ingredients. Traditionally, enzymes are isolated from cultivable microorganisms such as E. coli., or plants, and mammalian tissues, and are often not well-adapted to the modern food production. The use of recombinant DNA (rDNA) and vector technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. It is an urgent need in the field of application of biological enzymatic products for replacing any of the current potential harmful organic or synthesized chemical compounds for our public health.
For example, commonly used food sweeteners such as glucose or fructose syrups are typically produced from cornstarch using hydrolytic enzymes. In the first step of starch hydrolysis, starch is liquefied with α-amylase by heating at 105° C. for 2-5 min followed by 1-2 h at 90-100° C. With the advance of rDNA technology, it became possible to engineer amylases with increased heat stability and improved compatibility with other parameters of the liquefaction process. These improvements were accomplished by introducing changes in the α-amylase amino acid sequences through DNA sequence modifications of the α-amylase genes. Other enzymes currently used in food processing have also been improved using rDNA techniques.
The enzymes suitable for industrial application or other applications may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available (Table 1).
Enzymes produced by this vector system are also very useful in the fragrance/perfume industry. Chemical reagents have been used to produce scent compound to generate perfume with similar scent as those in nature counterparts. However, using chemical catalysis to produce active ingredients often produce both active form and its inactive twin form of molecules, as well as chemical reagents may remain as contamination in the final products. Enzyme is good to make only one of the versions to increase the purity of the final product without chemical toxicity. The enzyme used in the process is natural and scent produced is an exact replica of what is found in nature (i.e., in plant or animal); it thus can be considered as natural and health (perfume) products
TABLE 1Enzymes from recombinant microorganisms (based on FDA regulations,GRAS affirmation petitions, and GRAS notices)Source microorganismEnzymesReference*Aspergillus nigerPhytaseGRASP 2G0381Chymosin21 CFR 184.1685LipaseGRN 158Aspergillus oxyzaeEsterase-lipaseGRASP 7G0323Aspartic proteinaseGRN 34Glucose oxidaseGRN 106LaccaseGRN 122LipaseGRN 43; GRN 75;GRN 103Pectin esteraseGRN 8Phospholipase A1GRN 142Bacillus licheniformisα-amylaseGRASP 0G0363;GRN 22; GRN 24;GRN 79PullulanaseGRN 72Bacillus subtilisα-acetolactate21 CFR 173.115decarboxylaseα-amylaseGRASP 4G0293;GRASP 7G0328Maltogenic amylaseGRASP 7G0326PullulanaseGRN 20Escherichia coli K-12Chymosin21 CFR 184.1685Fusarium venenatumXylanaseGRN 54KluyveromycesChymosin21 CFR 184.1685marxianus var. lactisPseudomonas fluorescensα-amylaseGRN 126Biovar 1Trichoderma reeseiPectin lyaseGRN 32Reference: Z. S. Olempska-Beer et al./Regulatory Toxicology and Pharmacology 45 (2006) 144-158
In addition, there's need for using vector expression system as an economic biological method for large-scale production of cosmetic proteins or enzymes such as collagen, lipase, or other proteins or peptides, which are ideal candidates in whitening, depigmenting and wound-repairing applications. For example, novel engineered collagens with optimized biochemical and physical properties can be produced using either mammalian cell-lines or transgenic animals (Table 2).
TABLE 2Comparison of the various recombinant expression systems for the production of collagenYieldExpression hostProtein expressed(μg/ml)AdvantagesDisadvantagesYeastproα1(III) + α- and>15High yield, inexpensiveNot secreted, low(Pichia pastoris)β-subunits of P4Hhydroxylysine contentInsect cellsproα1(III) + α- and60High yieldNot secretedβ-subunits of P4HHT1080proα1(II), proα1(I),035-2Secreted, authenticLow yieldsproα1(III)product, no need for co-expression of P4HHEKproα1(V)15High yields, secreted,Some cleavage of293-EBNAauthentic product, nopropeptidesneed for co-expressionof P4HTransgenicModified procollagens + α-150High yield, authenticHigh developmentanimalsand β-subunits of P4HproductcostsReference: Biochemical Society Transactions (2000) Volume 28, part 4
In the future, these recombinant proteins can be used both to investigate the molecular basis and biochemistry of collagen assembly and to produce collagens with new pharmaceutical and medical uses. Similarly, the vector expression system can be utilized to generate other gene-modified functional proteins, which have extensive application in skin repairing, healing and aging protection.
Vector expression system can also be used in stem cell and gene therapy. For example, Gaucher disease is a lysosomal storage disorder resulting from a deficiency of an enzyme, glucocerebrosidase (GC). Recently, lentivirus vectors have been developed for efficient gene transfer into hematopoietic stem cells (HSCs). A recombinant lentivirus vector was used to evaluate the transduction of the human GC gene into murine bone-marrow-derived HSCs and its expression in their progeny. The recombinant lentiviral vector transduces HSCs that are capable of long-term gene expression in vivo; which was described in US Patent Publication US20030119770 A1. In addition, expression or production of fusion protein or enzyme, such as TatNP22-GC with capability to cross the blood brain-barrier is designed and made by the vector system. This approach is potentially useful for the treatment of patients with Gaucher disease, CNS disorders and other diseases.
Another application is recent development of lentiviral vector. It is especially useful for studies on gene or genomic function because the lentiviral vector can be used to achieve efficient integration of transgene into nondividing cell genomes and successful long-term expression of the transgene. These attributes make the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. This technique should facilitate the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of reporter genes. Our findings suggest that the reporter gene is driven by an upstream, cell-specific promoter during cell culture and cell differentiation, which further supports the usefulness of lentivirus-based gene-trap vectors. Lentiviral gene-trap vectors appear to offer a wealth of possibilities for the study of cell differentiation and lineage commitment, as well as for the discovery of new genes, tacking the migration of gene products, and identifying markers for early-stage human cancer cells' progressing activity.
Implantation of the serotonergic-like progenitors into the hippocampus of adult mice genetically lacking SERT was followed by migration of these cells into adjacent brain regions, and survival of the cells for many months was accompanied by a gradual increase in density of SERT protein expression, which was not found in vehicle-injected, control mice. These findings suggest that this serotonergic-like NSC model will be a useful contribution to the development of cell biotechnology in regard to the expression of missing genes such as SERT in the adult brain by employing appropriate vectors.